WO2007057557A1

WO2007057557A1 - Biochip with an improved fluorescent signal/parasite signal ratio

Info

Publication number: WO2007057557A1
Application number: PCT/FR2006/002518
Authority: WO
Inventors: Stéphane Getin; Guillaume Delapierre; Philippe Peltie; Patrick Chaton
Original assignee: Commissariat A L'energie Atomique
Priority date: 2005-11-15
Filing date: 2006-11-14
Publication date: 2007-05-24
Also published as: FR2893415A1

Abstract

The invention relates to a biochip comprising a substrate (12) supporting at least one reflective thin layer (13) providing a reception face for chemical or biological elements containing at least one constituent that can emit a fluorescent signal under the action of an excitation signal. The reflective layer (13) has a reflection coefficient of less than 4 % for the wavelength of the excitation signal and a reflection coefficient higher than 40 % for the wavelength of the fluorescent signal. The reflective layer (13) introduces a phase difference which is essentially equal to 360° between the fluorescent signal emitted by the constituent and the fluorescent signal reflected by the reflective layer (13).

Description

Biochip with fluorescent signal ratio / improved parasitic signal

Technical field of the invention

The invention relates to a biochip comprising a substrate supporting at least one reflective thin layer providing a reception face for chemical or biological elements containing at least one component able to emit a fluorescent signal under the action of an excitation signal, said reflective layer having a reflection coefficient of less than 4% at the wavelength of the excitation signal and a reflection coefficient greater than 40% at the wavelength of the fluorescent signal.

State of the art

Fluorescent labeling devices are particularly used in areas based on the detection of chemical or biological reactions (for example DNA microarrays, protein chips, cell chips, sugar chips ...).

Such devices use the fluorescence phenomenon. This phenomenon occurs when a molecule emits, in the form of a fluorescent signal, a part of the energy stored by absorption of an excitation light signal coming from a light source such as a laser, a fluorescent lamp or a light source. arc or light-emitting diodes (LEDs). The fluorescent signal is most often in the form of ultraviolet or visible radiation having a longer wavelength than that of the excitation signal.

By way of illustration, a fluorescence imaging device 1 according to the prior art is shown in FIG. 1. The imaging device 1 operates according to the principle of epifluorescence. It has a light source 2 intended to emit an excitation signal 3 in the direction of a separator cube 4 in which the excitation signal 3 is reflected in the direction of chemical or biological elements 5 to be analyzed. When the excitation signal 3 reaches the elements 5, which are deposited on a substrate 6, the latter re-emit a fluorescent signal 7 after excitation of at least one fluorescent component contained in the elements 5. At the same time, the signal d excitation 3 is reflected on the upper face 8 of the substrate 6 where the elements 5 to be analyzed are deposited to create a reflected excitation signal 9, the intensity of which depends on the reflection coefficient of the upper face 8 of the substrate 6. coefficient is, for example, close to 4% in the case of a glass slide. The fluorescent signal 7 and the reflected excitation signal 9 pass through a filter 10 which essentially passes the fluorescent signal 7, which is then read by a detector 11, for example a CCD camera or a photomultiplier.

The detection of chemical or biological reactions on a substrate 6 by fluorescent labeling is made difficult by the low level of intensity of the fluorescent signal 7 emitted by the fluorescent constituents, generally fluorophores. US6177990-B1 describes an epifluorescence imaging system, similar to the imaging device 1 of Figure 1, but to overcome the above disadvantage by enhancing fluorescence. In this system, the substrate comprises a rigid base having a specular, smooth, generally flat reflecting surface, and a transparent coating layer deposited on said reflecting surface. The coating layer has a thickness selected so that the optical path from the top of the coating layer to the reflecting surface is one quarter of the wavelength of the excitation signal. The chemical or biological elements to be analyzed are deposited on the upper face of the coating layer. This system makes it possible to reinforce the excitation signal, which induces a concomitant increase in the fluorescent signal. This epifluorescence imaging system is not completely satisfactory. Indeed, although the fluorescent signal received by the detector is higher than in the prior art, the latter also receives a higher parasitic signal because of the enhancement of the excitation signal. In practice, the ratio between the fluorescent signal and the parasitic signal is lower than for a conventional glass slide, deteriorating the quality of the measured results. This makes, moreover, impossible the analysis of chemical or biological elements of very low concentrations because, in this case, the ratio between the fluorescent signal and the spurious signal is then too low. These disadvantages due to the constitution of the substrate are present regardless of the type of imaging device in which said substrate is used.

The document US2004 / 0092028-A1 describes a biochip enabling a fluorescence enhancement by means of a thin layer or a thin film stack providing a mirror function for the wavelength of the excitation signal. The thickness e of the thin layer or the thickness of each thin layer of the stack is calculated from the following formula:

N.e = k./4 where N is the refractive index of the thin film material for the length of the excitation signal and k is an odd integer. This type of biochip makes it possible to increase the fluorescent signal by a reinforcement of the excitation signal and thus presents the same disadvantages as the substrate described in the document US6177990-B1.

The document WO2004 / 109264 describes, for its part, a sample substrate comprising an interference multilayer coating having at least two layers, wherein the thickness of the layers ensures that the fluorescent signal emitted by the corresponding constituent placed on the upper face of this layer. coating is reflected. This document provides that the thickness of the layers guarantees a reflection of almost all (99.5%) of the light emitted by the fluorophores, as well as a transmission of almost all (98%) of the excitation light not absorbed by the fluorescent material. These substrates, however, do not provide satisfactory results.

Object of the invention

The object of the invention is to remedy these drawbacks and, more particularly, to improve the ratio between the fluorescent signal and the parasitic signal detected by a fluorescence imaging device.

This object is achieved by a biochip according to the appended claims. In particular, the present invention thus provides a biochip where the reflective layer introduces a phase difference substantially equal to 360 ° between the fluorescent signal emitted by the constituent and the fluorescent signal reflected by the reflective layer.

Brief description of the drawings

Other advantages and features will emerge more clearly from the following description of particular embodiments of the invention given by way of non-limiting example and represented in the accompanying drawings, in which: FIG. 1 illustrates a particular embodiment of a fluorescence imaging device according to the prior art, - Figure 2 is a cross-sectional view of a biochip according to the invention, Figure 3 is a cross-sectional view of an exemplary embodiment of biochip according to the invention, optimized for the fluorophore CY3, the reflective layer constituting a Fabry-Perot cavity, FIG. 4 illustrates the evolution of the reflection coefficient of the reflective layer of the biochip of FIG. 3, as a function of The wavelength an incident signal,

FIG. 5 illustrates the parasitic signal measured for three biochips, respectively according to the prior art (A and B) and according to the invention (C), FIG. 6 illustrates the ratio K between the measured fluorescent signal and the measured parasite signal. for the three biochips A, B and C, Figure 7 shows the brightness received by the detector for each of the three biochips A, B and C.

Description of particular embodiments

Figure 2 is a cross-sectional view of a biochip according to the present invention. It represents a substrate 12 supporting a thin reflective layer 13. The latter provides a receiving face 14 for chemical or biological elements 15, schematized in the form of a layer. Said elements contain one or more fluorescent constituents intended to be activated by an excitation signal to emit a fluorescent signal (not shown in FIG. 2).

The thin reflective layer 13 has a reflection coefficient of less than 4% at the wavelength of the excitation signal and a reflection coefficient greater than 40% at the wavelength of the fluorescent signal. Thus, the reflected excitation signal is very weak, while the reflected fluorescence signal is important. Thus, while in the aforementioned prior art, certain biochips allow a fluorescence enhancement by an increase, by reflection, of the excitation signal, the biochip of FIG. 2 makes it possible to reinforce the fluorescence only thanks to a high reflection coefficient. (at least

40%) for the fluorescent signal. On the other hand, the reflected excitation signal is reduced as much as possible thanks to a reflection coefficient of less than 4% at this wavelength.

The thin reflective layer 13 is preferably constituted by a stacking thin sub-layers of dielectric materials alternately having a strong and low refractive index. These materials can be deposited by physical vapor deposition (PVD) type techniques, by chemical vapor deposition (CVD) or by sol-gel deposition (soaking / shrinking or centrifugation). . The thickness of the sub-layers can vary from a few tens to a few hundred nm.

The material with a high refractive index is for example chosen from the following materials: TiO ₂ , HfO ₂ , Si ₃ N ₄ , Ta ₂ O ₅ , Al ₂ O ₃ , In ₂ O ₃ . The material with a low refractive index is, for its part, chosen from, for example, the following materials: SiO ₂ , MgF ₂ , LiF.

Depending on the applications, the reflective layer 13, preferably in the form of a stack of thin sub-layers, may be deposited on the whole of the substrate 12 or on a structured substrate. In addition, the last sublayer must have a biological or chemical compatibility with the probes to be grafted on this underlayer. In general, the receiving face 14 must be chemically reactive for a bond with a given constituent of the chemical or biological elements 15, or must have electrostatic properties capable of creating a bond with said constituent.

Whatever the type of imaging device in which the biochip of FIG. 2 is used, the materials constituting the thin sub-layers, the thickness and the number of said sub-layers, are determined in such a way that the reflective layer 13 has a reflection coefficient of less than 4% at the wavelength of the excitation signal and a reflection coefficient greater than 40% at the wavelength of the fluorescent signal emitted by the elements 15.

A very high reflection coefficient of the layer 13 for the wavelength of the fluorescent signals is certainly an important parameter for the control of the emission of fluorescence, but it is essential to consider also the undulatory aspect of these signals. The signals emitted by the elements 15 can be decomposed into plane waves. Considering a single plane wave direction, defined by an angle measured with respect to the normal to the layer 13, the total signal received by the detector 11 corresponds to the sum of the intensity of the plane wave emitted by the elements. 15 and received directly by the detector 11 and the intensity of the plane wave reflected by the layer 15 and received, after reflection, by the detector 11. This sum results from the interference between these two plane waves and the intensity issued in the direction is written:

I = E _{1 +} E; I 2

where E ₁ and E ₂ represent the electromagnetic field respectively of the plane wave emitted by the elements 15 and received directly by the detector 11 and the plane wave reflected by the layer 15 and subsequently received by the detector 11.

And:

E ₁ = E ₀

E ₀ being the amplitude of the electromagnetic field emitted by the elements 15 in the direction.

In addition: E ₂ = E ₀ r exp (iφ)

where r is the reflection coefficient of the layer 13 at the wavelength of the fluorescent signals and φ the phase induced by the layer 13.

The intensity emitted in the direction is thus written: l = l _o | 1 + r exp (iφ) | ²

Consequently, the total intensity of the fluorescent signals received by the detector 11 is maximum for the following conditions: a maximum reflection coefficient of the fluorescent signals, for example close to 100%, a phase difference equal to 360 ° introduced by the layer 13 during the reflection of the fluorescent signals.

Thus, whatever the type of imaging device in which the biochip of FIG. 2 is used, the materials constituting the thin sub-layers, the thickness and the number of said sub-layers, are determined in such a way that the reflective layer 13 introduces a phase difference substantially equal to 360 ° between the fluorescent signals emitted by the elements 15 and the fluorescent signals reflected by the layer 13. In fact, the elements 15 emit fluorescent signals both in the direction of the filter 10 and of the detector 11, in the direction of the reflective layer 13. This phase difference eliminates any interference that might cancel the fluorescent signal emitted directly. With a phase shift of 360 °, the waves of the fluorescent signals coming directly to the detector 11 are in constructive interference with the waves of the fluorescent signals reflected on the layer 13.

By way of example, the wavelength of the excitation signal is 532 nm and that of the fluorescent signal is approximately 580 nm for the fluorophore CY3. Figure 3 illustrates an optimized biochip for this fluorophore. On the substrate 12, for example silicon, the reflective thin layer 13 has been deposited in the form of a stack of ten thin sub-layers 16 to 25. The sub-layers are alternately silica and titanium dioxide. Thus the sublayers 16, 18, 20, 22, and 24 are silica, of low refractive index, for example equal to 1.5 for the wavelength of the excitation signal. Money- layers 17, 19, 21, 23, and 25 are made of titanium dioxide TiO ₂ , of high refractive index, for example equal to 2.3 for the wavelength of the excitation signal. The sub-layers 16 to 25 are deposited with the PVD technique and their thicknesses are optical wavelengths for the excitation wavelength. The stack of sub-layers 16 to 25 constitutes a Fabry-Pérot cavity centered on the wavelength of the excitation signal.

FIG. 4 illustrates the evolution of the reflection coefficient CR of the reflective layer 13 of FIG. 3 as a function of the wavelength of an incident signal. According to the invention, the reflection coefficient CR is almost zero for the excitation wavelength (532nm). In contrast, CR is close to 100% at the wavelength of the fluorescent signal to enhance fluorescence.

Tests and measurements were performed with a second example of a biochip according to the invention (not shown). The latter comprises a substrate supporting a thin reflective layer consisting of a stack of 14 thin sub-layers of SiO ₂ alternated with 13 thin sub-layers of TiO ₂ . The following table shows the thickness of the underlayments:

In the tests, three biochips were compared: a conventional glass slide (referenced A), a biochip according to the teachings of the document

US2004 / 0092028-A1 (referenced B), the second example of a biochip according to the invention (referenced C), comprising the 27 thin sub-layers above.

The receiving face of the three biochips was prepared to be chemically reactive for the grafting of biological probes each containing fluorophores CY3.

The tests and measurements were carried out thanks to a scanner. FIG. 5 illustrates the parasitic signal measured for the three biochips and reveals on the one hand its increase with biochip B (from 55 to 113 NG) and on the other hand its decrease with biochip C (from 55 to 40 NG), by comparison with the biochip A. Figure 6 illustrates, for its part, the K ratio between the measured fluorescent signal and the measured parasitic signal. Figure 6 reveals that K decreases with biochip B (from 882 to 459) and increases with biochip C (from 882 to 1222), compared with biochip A.

FIG. 7 schematizes the intensity L of the optical signals received near a biological probe by the scanner detector for each of the three biochips A, B and C. FIG. 7 illustrates the intensity L, on the one hand, of the measured fluorescent signal (upper part of the slot) and, on the other hand, the measured parasitic signal (lower part of the slot). FIG. 7 confirms that the fluorescent signal measured for case C is clearly superior to the fluorescent signal of case A, thanks to the reflection coefficient of the reflective thin layer, which is greater than 40% for the wavelength of the fluorescent signal. For case A, the reflection coefficient is approximately equal to 4% irrespective of the length of the incident wave. In addition, the parasitic signal in case C is less than or equal to that of case A due to a reflection coefficient of the reflective layer of less than 4%, for the excitation signal. The ratio between the fluorescent signal and the parasitic signal is therefore significantly improved for case C compared to biochip A.

In addition, the reflection coefficient of the reflective thin film at the wavelength of the excitation signal is significantly lower in case C (equal to less than 4%) than in case B (at least 60%). Since the intensity of the reflected excitation signal varies according to the value of this coefficient, the reflected excitation signal is significantly weaker in case C. However, the signal parasite measured increases with the reflected excitation signal. Indeed, in practice, a part of the reflected excitation signal passes through the filter 10 placed in front of the detector 11 and the filter 10 itself can be made fluorescent at this irradiation. Therefore, and as shown in Figure 7, the spurious signal in case B is much higher than in case C.

On the other hand, the fluorescence signal received from the biochip C is lower than that received from the biochip B, because, with this, the increase of the excitation signal increases the emission of the detected fluorescent signal. However, the reduction of the fluorescent signal detected is less important than that of the spurious signal, because the reflection coefficient of the thin layer reflecting at the wavelength of the fluorescent signal remains high (at least 40%) in the biochip C. The The ratio between the fluorescent signal and the parasitic signal is therefore improved with the biochip C.

Increasing the ratio between the fluorescent signal and the parasitic signal by means of the biochip in accordance with the invention makes it possible to improve the quality of analysis. In particular, this makes possible the analysis of chemical or biological elements of very low concentrations by means of an adequate amplification of the signals received by the detector.

Finally, whatever the type of imaging device in which the biochip according to the invention is used, the materials constituting the thin sub-layers of the reflecting layer, the thickness and the number of said sub-layers are determined. by any appropriate means, for example by means of experimental results or numerical calculations. In particular, it is possible to use known software or optimization methods, for example from the article by AV Tikhonravov and the "Application of the needle optimization technique to the design of optical coatings" (Applied Optics - October 1, 1996 - Vol 35 - N 28).

Claims

claims

A biochip comprising a substrate (12) supporting at least one thin reflective layer (13) having a receiving face (14) for chemical or biological elements (15) containing at least one component capable of emitting a fluorescent signal (7) under the action of an excitation signal (3), said reflective layer (13) having a reflection coefficient of less than 4% at the wavelength of the excitation signal (3) and a higher reflection coefficient at 40% at the wavelength of the fluorescent signal (7), a biochip characterized in that said reflecting layer (13) introduces a phase difference substantially equal to 360 ° between the fluorescent signal emitted by the constituent and the reflected fluorescent signal by the reflective layer (13).

2. biochip according to claim 1, characterized in that the reflective thin layer (13) is constituted by a stack of thin sub-layers (16 to 25) of materials alternately having a strong refractive index and low.

3. Biochip according to claim 2, characterized in that said stack constitutes a Fabry-Pérot cavity centered on the wavelength of the excitation signal (3).

4. biochip according to one of claims 2 and 3, characterized in that the material with high refractive index is selected from the following materials: TiO ₂ , HfO ₂ , Si ₃ N ₄ , Ta ₂ O ₅ , Al ₂ O ₃ , In ₂ O ₃ .

5. Biochip according to any one of claims 2 to 4, characterized in that the material with a low refractive index is chosen from following materials: SiO ₂ , MgF ₂ , LiF.

6. biochip according to any one of claims 1 to 5, characterized in that the receiving face (14) of the thin reflecting layer (13) is chemically reactive for binding with a constituent of the chemical or biological elements (15). .

7. biochip according to any one of claims 1 to 5, characterized in that the receiving face (14) of the reflective layer (13) has electrostatic properties capable of creating an attachment of a constituent of the chemical or biological elements. (15).